Thin-Layer-Agar-Based Direct Phenotypic Drug Susceptibility Testing on Sputum in Eswatini Rapidly Detects Mycobacterium tuberculosis Growth and Rifampicin Resistance Otherwise Missed by WHO-Endorsed Diagnostic Tests.

Xpert MTB/RIF rapidly detects resistance to rifampicin (RR); however, this test misses I491F-RR conferring rpoB mutation, common in southern Africa. In addition, Xpert MTB/RIF does not distinguish between viable and dead Mycobacterium tuberculosis (MTB). We aimed to investigate the ability of thin-layer agar (TLA) direct drug-susceptibility testing (DST) to detect MTB and its drug-resistance profiles in field conditions in Eswatini. Consecutive samples were tested in parallel with Xpert MTB/RIF and TLA for rifampicin (1.0 µg/ml) and ofloxacin (2.0 µg/ml). TLA results were compared at the Reference Laboratory in Antwerp with indirect-DST on Löwenstein-Jensen or 7H11 solid media and additional phenotypic and genotypic testing to resolve discordance. TLA showed a positivity rate for MTB detection of 7.1% versus 10.0% for Xpert MTB/RIF. Of a total of 4,547 samples included in the study, 200 isolates were available for comparison to the composite reference. Within a median of 18.4 days, TLA detected RR with 93.0% sensitivity (95% confidence interval [CI], 77.4 to 98.0) and 99.4% specificity (95% CI, 96.7 to 99.9) versus 62.5% (95% CI, 42.7 to 78.8) and 99.3% (95% CI, 96.2 to 99.9) for Xpert MTB/RIF. Eight isolates, 28.6% of all RR-confirmed isolates, carried the I491F mutation, all detected by TLA. TLA also correctly identified 183 of the 184 ofloxacin-susceptible isolates (99.5% specificity; 95% CI, 97.0 to 99.9). In field conditions, TLA rapidly detects RR, and in this specific setting, it contributed to detection of additional RR patients over Xpert MTB/RIF, mainly but not exclusively due to I491F. TLA also accurately excluded fluoroquinolone resistance.

Characterization of Genomic Variants Associated with Resistance to Bedaquiline and Delamanid in Naive Mycobacterium tuberculosis Clinical Strains.

The role of mutations in genes associated with phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly characterized. A clear understanding of the genetic variants' role is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analyzed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole-genome sequencing (WGS) data set from a collection of 4,795 MTBc clinical isolates from six countries with a high burden of tuberculosis (TB). From WGS analysis, we identified 61 and 163 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to these medicines. To characterize the role of mutations, we calculated the free energy variation upon mutations in the available protein structures of Ddn (DLM), Fgd1 (DLM), and Rv0678 (BDQ) and performed MIC assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural and phenotypic data allowed for cataloguing the mutations clearly associated with resistance to BDQ (n = 4) and DLM (n = 35), only two of which were previously described, as well as about a hundred genetic variants without any correlation with resistance. Significantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance for the development of comprehensive molecular diagnostic tools.

Perspectives for personalized therapy for patients with multidrug-resistant tuberculosis.

According to the World Health Organization (WHO), tuberculosis is the leading cause of death attributed to a single microbial pathogen worldwide. In addition to the large number of patients affected by tuberculosis, the emergence of Mycobacterium tuberculosis drug-resistance is complicating tuberculosis control in many high-burden countries. During the past 5 years, the global number of patients identified with multidrug-resistant tuberculosis (MDR-TB), defined as bacillary resistance at least against rifampicin and isoniazid, the two most active drugs in a treatment regimen, has increased by more than 20% annually. Today we experience a historical peak in the number of patients affected by MDR-TB. The management of MDR-TB is characterized by delayed diagnosis, uncertainty of the extent of bacillary drug-resistance, imprecise standardized drug regimens and dosages, very long duration of therapy and high frequency of adverse events which all translate into a poor prognosis for many of the affected patients. Major scientific and technological advances in recent years provide new perspectives through treatment regimens tailor-made to individual needs. Where available, such personalized treatment has major implications on the treatment outcomes of patients with MDR-TB. The challenge now is to bring these adances to those patients that need them most.

S. aureus endocarditis: Clinical aspects and experimental approaches.

Infective endocarditis (IE) is a life-threatening disease, caused by septic vegetations and inflammatory foci on the surface of the endothelium and the valves. Due to its complex and often indecisive presentation the mortality rate is still about 30%. Most frequently bacterial microorganisms entering the bloodstream are the underlying origin of the intracardiac infection. While the disease was primarily restricted to younger patients suffering from rheumatic heart streptococci infections, new at risk categories for Staphylococcus (S.) aureus infections arose over the last years. Rising patient age, increasing drug resistance, intensive treatment conditions such as renal hemodialysis, immunosuppression and long term indwelling central venous catheters but also the application of modern cardiac device implants and valve prosthesis have led to emerging incidences of S. aureus IE in health care settings and community. The aetiologic change has impact on the pathophysiology of IE, the clinical presentation and the overall patient management. Despite intensive research on appropriate in vitro and in vivo models of IE and gained knowledge about the fundamental mechanisms in the formation of bacterial vegetations and extracardiac complications, improved understanding of relevant bacterial virulence factors and triggered host immune responses is required to help developing novel antipathogenic treatment strategies and pathogen specific diagnostic markers. In this review, we summarize and discuss the two main areas affected by the changing patient demographics and provide first, recent knowledge about the pathogenic strategies of S. aureus in the induction of IE, including available experimental models of IE used to study host-pathogen interactions and diagnostic and therapeutic targets. In a second focus we present diagnostic (imaging) regimens for patients with S. aureus IE according to current guidelines as well as treatment strategies and surgical recommendations.

Direct DNA Extraction from Mycobacterium tuberculosis Frozen Stocks as a Reculture-Independent Approach to Whole-Genome Sequencing.

Culturing before DNA extraction represents a major time-consuming step in whole-genome sequencing of slow-growing bacteria, such as Mycobacterium tuberculosis. We report a workflow to extract DNA from frozen isolates without reculturing. Prepared libraries and sequence data were comparable with results from recultured aliquots of the same stocks.

Mutations in SDHC cause autosomal dominant paraganglioma, type 3.

Nonchromaffin paragangliomas (PGLs) are usually benign, neural-crest-derived, slow-growing tumours of parasympathetic ganglia. Between 10% and 50% of cases are familial and are transmitted as autosomal dominant traits with incomplete and age-dependent penetrance.

Performance of spirometry assessment at TB diagnosis.

BACKGROUND: Spirometry is considered relevant for the diagnosis and monitoring of post-TB lung disease. However, spirometry is rarely done in newly diagnosed TB patients.METHODS: Newly diagnosed, microbiologically confirmed TB patients were recruited for the study. Spirometry was performed within 21 days of TB treatment initiation according to American Thoracic Society/European Respiratory Society guidelines. Spirometry analysis was done using Global Lung Initiative equations for standardisation.RESULTS: Of 1,430 eligible study participants, 24.7% (353/1,430) had no spirometry performed mainly due to contraindications and 23.0% (329/1,430) had invalid results; 52.3% (748/1,430) of participants had a valid result, 82.8% (619/748) of whom had abnormal spirometry. Of participants with abnormal spirometry, 70% (436/619) had low forced vital capacity (FVC), 6.1% (38/619) had a low ratio of forced expiratory volume in 1 sec (FEV1) to FVC, and 19.1% (118/619) had low FVC, as well as low FEV1/FVC ratio. Among those with abnormal spirometry, 26.3% (163/619) had severe lung impairment.CONCLUSIONS: In this population, a high proportion of not performed and invalid spirometry assessments was observed; this was addressed by removing tachycardia as a (relative) contraindication from the study guidance and retraining. The high proportion of patients with severe pulmonary impairment at the time of TB diagnosis suggests a huge morbidity burden and calls for further longitudinal studies on the relevance of spirometry in predicting chronic lung impairment after TB.

[Tuberculosis outbreak in 13 people in Saxony-Anhalt: indications of an infection chain by spoligotyping]. / Tuberkulose-Ausbruch bei 13 Personen in Sachsen-Anhalt: Hinweis auf die Infektkette durch Spoligotyping.

BACKGROUND: In Germany the number of new tuberculosis incidents continues to decline slightly. Cases of illnesses are often diagnosed late, due to the uncharacteristic course of tuberculosis. The success in environment investigations by health authorities in finding incidents within one infection chain depends on the compliance of patients among other factors. Tuberculosis occurs especially in groups with social disadvantages. In this population segment the identification of contact persons of a patient with infectious tuberculosis presents difficulties. Moreover, identifying the source of infection is frequently not successful. For these reasons, additional infections and sicknesses are caused. The cultural pathogen proof, including type differentiation and resistance testing, continues to be the "gold standard" in tuberculosis diagnostics and is required for the examination of outbreaks. Molecular methods for fine typification of isolated bacterial strains and their subsequent comparison constitute an important tool in the infection epidemiology investigation of incident clustering. OBJECTIVE AND METHOD: The objective of this work is the description of a tuberculosis infection chain in Saxony-Anhalt, which was discovered by means of a molecular biological method. The conservative method represents the environment investigations for the expedient selection of contact persons. The investigations always take 2 directions in the process. On the one hand, it is searched for the still unknown source of infection. On the other hand, persons are to be identified, who have been infected before the diagnosis became known. The diagnostics for contact persons includes besides the lung X-ray checkup exam, the Mendel Mantoux tuberculin skin test (THT), sputum tests and the Interferon Gamma (INF-γ) tests (IGRA). In described case, the molecular biological method of spoliogotyping was employed as additional instrument. Therein, fingerprint maps of the patients' tuberculosis strings were created and compared. Infection chains resulting from the environment investigations were thereby secured. RESULTS: We report on a tuberculosis outbreak in a district of the state of Saxony-Anhalt, which occurred in the time from June 2007 to May 2010. As result of the environment investigations, an infection chain of 13 ill patients (77% ♂, 23% ♀) was identified. In 11 cases the correlation of the infection chain was proven by spoligotyping (9 ♂, 2 ♀). 2 cases (1 ♂, 1 ♀) without verification of the pathogen cannot be attributed on basis of the epidemiological correlations.

Whole-Genome Sequencing for Resistance Prediction and Transmission Analysis of Mycobacterium tuberculosis Complex Strains from Namibia.

Namibia is among 30 countries with a high burden of tuberculosis (TB), with an estimated incidence of 460 per 100,000 population and around 800 new multidrug-resistant (MDR) TB cases per year. Still, data on the transmission and evolution of drug-resistant Mycobacterium tuberculosis complex (Mtbc) strains are not available. Whole-genome sequencing data of 136 rifampicin-resistant (RIFr) Mtbc strains obtained from 2016 to 2018 were used for phylogenetic classification, resistance prediction, and cluster analysis and linked with phenotypic drug susceptibility testing (pDST) data. Roughly 50% of the strains investigated were resistant to all first-line drugs. Furthermore, 13% of the MDR Mtbc strains were already pre-extensively drug resistant (pre-XDR). The cluster rates were high, at 74.6% among MDR and 85% among pre-XDR strains. A significant proportion of strains had borderline resistance-conferring mutations, e.g., inhA promoter mutations or rpoB L430P. Accordingly, 25% of the RIFr strains tested susceptible by pDST. Finally, we determined a potentially new bedaquiline resistance mutation (Rv0678 D88G) occurring in two independent clusters. High rates of resistance to first-line drugs in line with emerging pre-XDR and likely bedaquiline resistance linked with the ongoing recent transmission of MDR Mtbc clones underline the urgent need for the implementation of interventions that allow rapid diagnostics to break MDR TB transmission chains in the country. A borderline RIFr mutation in the dominant outbreak strain causing discrepancies between phenotypic and genotypic resistance testing results may require breakpoint adjustments but also may allow individualized regimens with high-dose treatment. IMPORTANCE The transmission of drug-resistant tuberculosis (TB) is a major problem for global TB control. Using genome sequencing, we showed that 13% of the multidrug-resistant (MDR) M. tuberculosis complex strains from Namibia are already pre-extensively drug resistant (pre-XDR), which is substantial in an African setting. Our data also indicate that the ongoing transmission of MDR and pre-XDR strains contributes significantly to the problem. In contrast to other settings with higher rates of drug resistance, we found a high proportion of strains having so-called borderline low-level resistance mutations, e.g., inhA promoter mutations or rpoB L430P. This led to the misclassification of 25% of the rifampicin-resistant strains as susceptible by phenotypic drug susceptibility testing. This observation potentially allows individualized regimens with high-dose treatment as a potential option for patients with few treatment options. We also found a potentially new bedaquiline resistance mutation in rv0678.

Library preparation for next generation sequencing: A review of automation strategies.

Next generation sequencing is in the process of evolving from a technology used for research purposes to one which is applied in clinical diagnostics. Recently introduced high throughput and benchtop instruments offer fully automated sequencing runs at a lower cost per base and faster assay times. In turn, the complex and cumbersome library preparation, starting with isolated nucleic acids and resulting in amplified and barcoded DNA with sequencing adapters, has been identified as a significant bottleneck. Library preparation protocols usually consist of a multistep process and require costly reagents and substantial hands-on-time. Considerable emphasis will need to be placed on standardisation to ensure robustness and reproducibility. This review presents an overview of the current state of automation of library preparation for next generation sequencing. Major challenges associated with library preparation are outlined and different automation strategies are classified according to their functional principle. Pipetting workstations allow high-throughput processing yet offer limited flexibility, whereas microfluidic solutions offer great potential due to miniaturisation and decreased investment costs. For the emerging field of single cell transcriptomics for example, microfluidics enable singularisation of tens of thousands of cells in nanolitre droplets and barcoding of the RNA to assign each nucleic acid sequence to its cell of origin. Finally, two applications, the characterisation of bacterial pathogens and the sequencing within human immunogenetics, are outlined and benefits of automation are discussed.

International Society of Cardiovascular Infectious Diseases Guidelines for the Diagnosis, Treatment and Prevention of Disseminated Mycobacterium chimaera Infection Following Cardiac Surgery with Cardiopulmonary Bypass.

Mycobacterial infection-related morbidity and mortality in patients following cardiopulmonary bypass surgery is high and there is a growing need for a consensus-based expert opinion to provide international guidance for diagnosing, preventing and treating in these patients. In this document the International Society for Cardiovascular Infectious Diseases (ISCVID) covers aspects of prevention (field of hospital epidemiology), clinical management (infectious disease specialists, cardiac surgeons, ophthalmologists, others), laboratory diagnostics (microbiologists, molecular diagnostics), device management (perfusionists, cardiac surgeons) and public health aspects.

Multicentre study to establish interpretive criteria for clofazimine drug susceptibility testing.

To conduct a multicentre study to establish the critical concentration (CC) for clofazimine (CFZ) for drug susceptibility testing (DST) of Mycobacterium tuberculosis on the MGIT™960™ system using the distribution of minimum inhibitory concentrations (MIC) and genotypic analyses of Rv0678 mutations. In phase I of the study, the MIC distribution of laboratory strains (H37Rv and in vitro-selected Rv0678 mutants) and clinical pan-susceptible isolates were determined (n = 70). In phase II, a tentative CC for CFZ (n = 55) was proposed. In phase III, the proposed CC was validated using clinical drug-resistant tuberculosis (DR-TB) isolates stratified by Rv0678 mutation (n = 85). The MIC distribution of CFZ for laboratory and clinical pan-susceptible strains ranged between 0.125 µg/ml and 0.5 µg/ml. As the MIC values of DR-TB isolates used for phase II ranged between 0.25 µg/ml and 1 µg/ml, a CC of 1 µg/ml was proposed. Validation of the CC in phase III showed that probably susceptible and probably resistant Rv0678 mutants overlapped at 1 µg/ml. We therefore recommend a CC of 1 µg/ml, with additional testing at 0.5 µg/ml to define an intermediate category. This was the first comprehensive study to establish a CC for routine phenotypic DST of CFZ using the MGIT960 system to guide therapeutic decisions. .

Role and value of whole genome sequencing in studying tuberculosis transmission.

BACKGROUND: Tuberculosis (TB) remains a serious public health threat worldwide. Theoretically ultimate resolution of whole genome sequencing (WGS) for Mycobacterium tuberculosis complex (MTBC) strain classification makes this technology very attractive for epidemiological investigations. OBJECTIVES: To summarize the evidence available in peer-reviewed publications on the role and place of WGS in detection of TB transmission. SOURCES: A total of 69 peer-reviewed publications identified in Pubmed database. CONTENT: Evidence from >30 publications suggests that a cut-off value of fewer than six single nucleotide polymorphisms between strains efficiently excludes cases that are not the result of recent transmission and could be used for the identification of drug-sensitive isolates involved in direct human-to-human TB transmission. Sensitivity of WGS to identify epidemiologically linked isolates is high, reaching 100% in eight studies with specificity (17%-95%) highly dependent on the settings. Drug resistance and specific phylogenetic lineages may be associated with accelerated mutation rates affecting genetic distances. WGS can be potentially used to distinguish between true relapses and re-infections but in high-incidence low-diversity settings this would require consideration of epidemiological links and minority alleles. Data from four studies looking into within-host diversity highlight a need for developing criteria for acceptance or rejection of WGS relatedness results depending on the proportion of minority alleles. IMPLICATIONS: WGS will potentially allow for more targeted public health actions preventing unnecessary investigations of false clusters. Consensus on standardization of raw data quality control processing criteria, analytical pipelines and reporting language is yet to be reached.

In vivo virulence of Mycobacterium tuberculosis depends on a single homologue of the LytR-CpsA-Psr proteins.

LytR-cpsA-Psr (LCP) domain containing proteins fulfil important functions in bacterial cell wall synthesis. In Mycobacterium tuberculosis complex (Mtbc) strains, the causative agents of tuberculosis (TB), the genes Rv3484 and Rv3267 encode for LCP proteins which are putatively involved in arabinogalactan transfer to peptidoglycan. To evaluate the significance of Rv3484 for Mtbc virulence, we generated a deletion mutant in the Mtbc strain H37Rv and studied its survival in mice upon aerosol infection. The deletion mutant failed to establish infection demonstrating that Rv3484 is essential for growth in mice. Following an initial phase of marginal replication in the lungs until day 21, the Rv3484 deletion mutant was almost eliminated by day 180 post-infectionem. Interestingly, the mutant also showed higher levels of resistance to meropenem/clavulanate and lysozyme, both targeting peptidoglycan structure. We conclude that Rv3484 is essential for Mtbc virulence in vivo where its loss of function cannot be compensated by Rv3267.

Fast update of undetected Mycobacterium chimaera infections to reveal unsuspected cases.

Mycobacterium chimaera is involved in a worldwide alert due to contaminated heater-cooler units. A real-time polymerase chain reaction (RT-PCR)-based procedure was implemented to survey undetected cases of M. chimaera infection. PCR was negative in the 59 prosthetic heart valves from patients with PCR-16SrRNA-negative infective endocarditis. PCR identified M. chimaera in one of 15 clinically significant retrospective Mycobacterium avium-Mycobacterium intracellulare complex isolates, which corresponded to a patient who had undergone heart valve replacement in a different institution. Whole-genome sequencing demonstrated that he was the first case in Spain with involvement of the strain responsible for the global outbreak. These results highlight the relevance of retrospective tracking for undetected M. chimaera infections.

No associations of human pulmonary tuberculosis with Sp110 variants.

BACKGROUND: After a recent report on the role of the Ipr1 gene in mediating innate immunity in a mouse model of Mycobacterium tuberculosis infection, the human Ipr1 homologue, Sp110, was considered a promising candidate for an association study in human tuberculosis. METHODS: In a sample of >1000 sputum positive, HIV negative West African patients with pulmonary tuberculosis and >1000 exposed, apparently healthy controls, we have genotyped 21 Sp110 gene variants that were either available from public databases, including HapMap data, or identified by DNA re-sequencing. RESULTS: No significant differences in the frequencies of any of the 21 variants were observed between patients and controls. This applied also for HapMap tagging variants and the corresponding haplotypes, when including sliding window analyses with three adjacent variants, and when stratifying controls for positivity and negativity according to the results of intradermal tuberculin (purified protein derivative, PPD) skin tests. DNA re-sequencing revealed 13 novel Sp110 variants in the 5'-UTR, exons, and adjacent intronic regions. CONCLUSIONS: Based on the results obtained in this case-control study, the hypothesis that Sp110 variants and haplotypes might be associated with distinct phenotypes of human M tuberculosis infection is doubtful.

Novel rapid PCR for the detection of Ile491Phe rpoB mutation of Mycobacterium tuberculosis, a rifampicin-resistance-conferring mutation undetected by commercial assays.

OBJECTIVES: Neither the liquid medium-based Bactec MGIT, nor commercial molecular assays such as the Xpert MTB/RIF and the MTBDRplus V2.0 assays are capable of detecting up to 30% of rifampicin-resistant Mycobacterium tuberculosis strains in Swaziland because of the large proportion of the rpoB Ile491Phe mutations. In other countries, the frequency of this mutation is thought to be low. METHODS: We designed a real-time multiplex allele-specific PCR assay to identify the rpoB Ile491Phe mutation responsible for these undetected resistant M. tuberculosis strains. RESULTS: The technique showed 100% similarity with rpoB sequencing on a panel of 78 strains from Swaziland. CONCLUSIONS: We propose that the detection of the rpoB Ile491Phe rpoB mutation should complement commercial assays for the diagnosis of rifampicin-resistant M. tuberculosis in routine conditions, particularly in countries where this specific mutation is frequent. The technique proposed in this paper is adapted for most reference laboratories.

Whole genome sequencing for M/XDR tuberculosis surveillance and for resistance testing.

Whole genome sequencing (WGS) can help to relate Mycobacterium tuberculosis genomes to one another to assess genetic relatedness and infer the likelihood of transmission between cases. The same sequence data are now increasingly being used to predict drug resistance and susceptibility. Controlling the spread of tuberculosis and providing patients with the correct treatment are central to the World Health Organization's target to 'End TB' by 2035, for which the global prevalence of drug-resistant tuberculosis remains one of the main obstacles to success. So far, WGS has been applied largely to drug-susceptible strains for the purposes of understanding transmission, leaving a number of analytical considerations before transferring what has been learnt from drug-susceptible disease to drug-resistant tuberculosis. We discuss these potential problems here, alongside some of the challenges to characterizing the Mycobacterium tuberculosis 'resistome'-the optimal knowledge-base required for WGS-based assays to successfully direct individualized treatment regimens through the prediction of drug resistance and susceptibility in the future.